Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1990 Nov;10(11):5707–5720. doi: 10.1128/mcb.10.11.5707

The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication.

S I Gibson 1, R T Surosky 1, B K Tye 1
PMCID: PMC361340  PMID: 2233713

Abstract

MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells. It encodes a protein of 971 amino acids that shows striking homology to the Mcm2 protein. We have mapped the mcm3-1 mutation of the left arm of chromosome V approximately 3 kb centromere proximal of anp1. The mcm3-1 mutant was found to be thermosensitive for growth. Under permissive growth conditions, it was defective in minichromosome maintenance in an autonomously replicating sequence-specific manner and showed an increase in chromosome loss and recombination. Under nonpermissive conditions, mcm3-1 exhibited a cell cycle arrest phenotype, arresting at the large-bud stage with an undivided nucleus that had a DNA content of nearly 2n. These phenotypes are consistent with incomplete replication of the genome of the mcm3-1 mutant, possibly as a result of limited replication initiation at selective autonomously replicating sequences leading to cell cycle arrest before mitosis. The phenotype exhibited by the mcm3 mutant is very similar to that of mcm2, suggesting that the Mcm2 and Mcm3 protein may play interacting roles in DNA replication.

Full text

PDF
5707

Images in this article

5711Image
on p.5711
5717Image
on p.5717
5717Image
on p.5717

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker T. A., Sekimizu K., Funnell B. E., Kornberg A. Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome. Cell. 1986 Apr 11;45(1):53–64. doi: 10.1016/0092-8674(86)90537-4. [DOI] [PubMed] [Google Scholar]
  2. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  3. Bouton A. H., Smith M. M. Fine-structure analysis of the DNA sequence requirements for autonomous replication of Saccharomyces cerevisiae plasmids. Mol Cell Biol. 1986 Jul;6(7):2354–2363. doi: 10.1128/mcb.6.7.2354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Brewer B. J., Fangman W. L. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell. 1987 Nov 6;51(3):463–471. doi: 10.1016/0092-8674(87)90642-8. [DOI] [PubMed] [Google Scholar]
  6. Broach J. R., Li Y. Y., Feldman J., Jayaram M., Abraham J., Nasmyth K. A., Hicks J. B. Localization and sequence analysis of yeast origins of DNA replication. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):1165–1173. doi: 10.1101/sqb.1983.047.01.132. [DOI] [PubMed] [Google Scholar]
  7. Broker T. R., Doermann A. H. Molecular and genetic recombination of bacteriophage T4. Annu Rev Genet. 1975;9:213–244. doi: 10.1146/annurev.ge.09.120175.001241. [DOI] [PubMed] [Google Scholar]
  8. Buchman A. R., Kimmerly W. J., Rine J., Kornberg R. D. Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jan;8(1):210–225. doi: 10.1128/mcb.8.1.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Celniker S. E., Sweder K., Srienc F., Bailey J. E., Campbell J. L. Deletion mutations affecting autonomously replicating sequence ARS1 of Saccharomyces cerevisiae. Mol Cell Biol. 1984 Nov;4(11):2455–2466. doi: 10.1128/mcb.4.11.2455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Conrad M. N., Newlon C. S. Saccharomyces cerevisiae cdc2 mutants fail to replicate approximately one-third of their nuclear genome. Mol Cell Biol. 1983 Jun;3(6):1000–1012. doi: 10.1128/mcb.3.6.1000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dale R. M., McClure B. A., Houchins J. P. A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA. Plasmid. 1985 Jan;13(1):31–40. doi: 10.1016/0147-619x(85)90053-8. [DOI] [PubMed] [Google Scholar]
  12. Dasso M., Newport J. W. Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus. Cell. 1990 Jun 1;61(5):811–823. doi: 10.1016/0092-8674(90)90191-g. [DOI] [PubMed] [Google Scholar]
  13. Dean F. B., Bullock P., Murakami Y., Wobbe C. R., Weissbach L., Hurwitz J. Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication. Proc Natl Acad Sci U S A. 1987 Jan;84(1):16–20. doi: 10.1073/pnas.84.1.16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dieckmann C. L., Tzagoloff A. Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J Biol Chem. 1985 Feb 10;260(3):1513–1520. [PubMed] [Google Scholar]
  15. Diffley J. F., Stillman B. Purification of a yeast protein that binds to origins of DNA replication and a transcriptional silencer. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2120–2124. doi: 10.1073/pnas.85.7.2120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Diffley J. F., Stillman B. Similarity between the transcriptional silencer binding proteins ABF1 and RAP1. Science. 1989 Nov 24;246(4933):1034–1038. doi: 10.1126/science.2511628. [DOI] [PubMed] [Google Scholar]
  17. Dodson M., Echols H., Wickner S., Alfano C., Mensa-Wilmot K., Gomes B., LeBowitz J., Roberts J. D., McMacken R. Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7638–7642. doi: 10.1073/pnas.83.20.7638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Eisenberg S., Civalier C., Tye B. K. Specific interaction between a Saccharomyces cerevisiae protein and a DNA element associated with certain autonomously replicating sequences. Proc Natl Acad Sci U S A. 1988 Feb;85(3):743–746. doi: 10.1073/pnas.85.3.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Francesconi S. C., Eisenberg S. Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences. Mol Cell Biol. 1989 Jul;9(7):2906–2913. doi: 10.1128/mcb.9.7.2906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Game J. C., Johnston L. H., von Borstel R. C. Enhanced mitotic recombination in a ligase-defective mutant of the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4589–4592. doi: 10.1073/pnas.76.9.4589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goff C. G., Moir D. T., Kohno T., Gravius T. C., Smith R. A., Yamasaki E., Taunton-Rigby A. Expression of calf prochymosin in Saccharomyces cerevisiae. Gene. 1984 Jan;27(1):35–46. doi: 10.1016/0378-1119(84)90236-1. [DOI] [PubMed] [Google Scholar]
  22. Hand R. Eucaryotic DNA: organization of the genome for replication. Cell. 1978 Oct;15(2):317–325. doi: 10.1016/0092-8674(78)90001-6. [DOI] [PubMed] [Google Scholar]
  23. Hartwell L. H., Smith D. Altered fidelity of mitotic chromosome transmission in cell cycle mutants of S. cerevisiae. Genetics. 1985 Jul;110(3):381–395. doi: 10.1093/genetics/110.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hartwell L. H. Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae. J Bacteriol. 1973 Sep;115(3):966–974. doi: 10.1128/jb.115.3.966-974.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hsiao C. L., Carbon J. High-frequency transformation of yeast by plasmids containing the cloned yeast ARG4 gene. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3829–3833. doi: 10.1073/pnas.76.8.3829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hu N., Messing J. The making of strand-specific M13 probes. Gene. 1982 Mar;17(3):271–277. doi: 10.1016/0378-1119(82)90143-3. [DOI] [PubMed] [Google Scholar]
  28. Huberman J. A., Spotila L. D., Nawotka K. A., el-Assouli S. M., Davis L. R. The in vivo replication origin of the yeast 2 microns plasmid. Cell. 1987 Nov 6;51(3):473–481. doi: 10.1016/0092-8674(87)90643-x. [DOI] [PubMed] [Google Scholar]
  29. Huberman J. A., Zhu J. G., Davis L. R., Newlon C. S. Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae. Nucleic Acids Res. 1988 Jul 25;16(14A):6373–6384. doi: 10.1093/nar/16.14.6373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hutter K. J., Eipel H. E. Flow cytometric determinations of cellular substances in algae, bacteria, moulds and yeasts. Antonie Van Leeuwenhoek. 1978;44(3-4):269–282. doi: 10.1007/BF00394305. [DOI] [PubMed] [Google Scholar]
  31. Johnson A. D., Herskowitz I. A repressor (MAT alpha 2 Product) and its operator control expression of a set of cell type specific genes in yeast. Cell. 1985 Aug;42(1):237–247. doi: 10.1016/s0092-8674(85)80119-7. [DOI] [PubMed] [Google Scholar]
  32. Kearsey S. Structural requirements for the function of a yeast chromosomal replicator. Cell. 1984 May;37(1):299–307. doi: 10.1016/0092-8674(84)90326-x. [DOI] [PubMed] [Google Scholar]
  33. Kimmerly W., Buchman A., Kornberg R., Rine J. Roles of two DNA-binding factors in replication, segregation and transcriptional repression mediated by a yeast silencer. EMBO J. 1988 Jul;7(7):2241–2253. doi: 10.1002/j.1460-2075.1988.tb03064.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kleid D. G., Yansura D., Small B., Dowbenko D., Moore D. M., Grubman M. J., McKercher P. D., Morgan D. O., Robertson B. H., Bachrach H. L. Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine. Science. 1981 Dec 4;214(4525):1125–1129. doi: 10.1126/science.6272395. [DOI] [PubMed] [Google Scholar]
  35. Konrad E. B. Method for the isolation of Escherichia coli mutants with enhanced recombination between chromosomal duplications. J Bacteriol. 1977 Apr;130(1):167–172. doi: 10.1128/jb.130.1.167-172.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Langford C. J., Gallwitz D. Evidence for an intron-contained sequence required for the splicing of yeast RNA polymerase II transcripts. Cell. 1983 Jun;33(2):519–527. doi: 10.1016/0092-8674(83)90433-6. [DOI] [PubMed] [Google Scholar]
  37. Maine G. T., Sinha P., Tye B. K. Mutants of S. cerevisiae defective in the maintenance of minichromosomes. Genetics. 1984 Mar;106(3):365–385. doi: 10.1093/genetics/106.3.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Maine G. T., Surosky R. T., Tye B. K. Isolation and characterization of the centromere from chromosome V (CEN5) of Saccharomyces cerevisiae. Mol Cell Biol. 1984 Jan;4(1):86–91. doi: 10.1128/mcb.4.1.86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. McKnight G. L., Cardillo T. S., Sherman F. An extensive deletion causing overproduction of yeast iso-2-cytochrome c. Cell. 1981 Aug;25(2):409–419. doi: 10.1016/0092-8674(81)90059-3. [DOI] [PubMed] [Google Scholar]
  40. Morrison D. A. Transformation in Escherichia coli: cryogenic preservation of competent cells. J Bacteriol. 1977 Oct;132(1):349–351. doi: 10.1128/jb.132.1.349-351.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mortimer R. K., Schild D. Genetic map of Saccharomyces cerevisiae. Microbiol Rev. 1980 Dec;44(4):519–571. doi: 10.1128/mr.44.4.519-571.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  43. Passmore S., Elble R., Tye B. K. A protein involved in minichromosome maintenance in yeast binds a transcriptional enhancer conserved in eukaryotes. Genes Dev. 1989 Jul;3(7):921–935. doi: 10.1101/gad.3.7.921. [DOI] [PubMed] [Google Scholar]
  44. Passmore S., Maine G. T., Elble R., Christ C., Tye B. K. Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MAT alpha cells. J Mol Biol. 1988 Dec 5;204(3):593–606. doi: 10.1016/0022-2836(88)90358-0. [DOI] [PubMed] [Google Scholar]
  45. Petes T. D., Newlon C. S. Structure of DNA in DNA replication mutants of yeast. Nature. 1974 Oct 18;251(5476):637–639. doi: 10.1038/251637a0. [DOI] [PubMed] [Google Scholar]
  46. Petes T. D., Williamson D. H. Fiber autoradiography of replicating yeast DNA. Exp Cell Res. 1975 Oct 1;95(1):103–110. doi: 10.1016/0014-4827(75)90614-x. [DOI] [PubMed] [Google Scholar]
  47. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  48. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  49. Saffer L. D., Miller O. L., Jr Electron microscopic study of Saccharomyces cerevisiae rDNA chromatin replication. Mol Cell Biol. 1986 Apr;6(4):1148–1157. doi: 10.1128/mcb.6.4.1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Seifert H. S., Chen E. Y., So M., Heffron F. Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1986 Feb;83(3):735–739. doi: 10.1073/pnas.83.3.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Shore D., Stillman D. J., Brand A. H., Nasmyth K. A. Identification of silencer binding proteins from yeast: possible roles in SIR control and DNA replication. EMBO J. 1987 Feb;6(2):461–467. doi: 10.1002/j.1460-2075.1987.tb04776.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sinha P., Chang V., Tye B. K. A mutant that affects the function of autonomously replicating sequences in yeast. J Mol Biol. 1986 Dec 20;192(4):805–814. doi: 10.1016/0022-2836(86)90030-6. [DOI] [PubMed] [Google Scholar]
  54. Sinha S., Ramaswamy R. On the dynamics of controlled metabolic network and cellular behaviour. Biosystems. 1987;20(4):341–354. doi: 10.1016/0303-2647(87)90052-9. [DOI] [PubMed] [Google Scholar]
  55. Sitney K. C., Budd M. E., Campbell J. L. DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene. Cell. 1989 Feb 24;56(4):599–605. doi: 10.1016/0092-8674(89)90582-5. [DOI] [PubMed] [Google Scholar]
  56. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  57. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Surosky R. T., Tye B. K. Construction of telocentric chromosomes in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2106–2110. doi: 10.1073/pnas.82.7.2106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Thrash-Bingham C., Fangman W. L. A yeast mutation that stabilizes a plasmid bearing a mutated ARS1 element. Mol Cell Biol. 1989 Feb;9(2):809–816. doi: 10.1128/mcb.9.2.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Tschumper G., Carbon J. Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. Gene. 1980 Jul;10(2):157–166. doi: 10.1016/0378-1119(80)90133-x. [DOI] [PubMed] [Google Scholar]
  63. Umek R. M., Kowalski D. The ease of DNA unwinding as a determinant of initiation at yeast replication origins. Cell. 1988 Feb 26;52(4):559–567. doi: 10.1016/0092-8674(88)90469-2. [DOI] [PubMed] [Google Scholar]
  64. Vaitukaitis J. L. Production of antisera with small doses of immunogen: multiple intradermal injections. Methods Enzymol. 1981;73(Pt B):46–52. doi: 10.1016/0076-6879(81)73055-6. [DOI] [PubMed] [Google Scholar]
  65. Walker S. S., Francesconi S. C., Eisenberg S. A DNA replication enhancer in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4665–4669. doi: 10.1073/pnas.87.12.4665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Walker S. S., Francesconi S. C., Tye B. K., Eisenberg S. The OBF1 protein and its DNA-binding site are important for the function of an autonomously replicating sequence in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Jul;9(7):2914–2921. doi: 10.1128/mcb.9.7.2914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Williams J. S., Eckdahl T. T., Anderson J. N. Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jul;8(7):2763–2769. doi: 10.1128/mcb.8.7.2763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Williamson D. H., Fennell D. J. The use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA. Methods Cell Biol. 1975;12:335–351. doi: 10.1016/s0091-679x(08)60963-2. [DOI] [PubMed] [Google Scholar]
  69. Yocum R. R., Hanley S., West R., Jr, Ptashne M. Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Oct;4(10):1985–1998. doi: 10.1128/mcb.4.10.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES